This invention relates to the expression of sedoheptulose 1,7 bisphosphatase (SBPase) in transgenic plants to increase or improve plant growth and development, yield, vigor, and distribution of carbon assimilates. Transgenic plants expressing SBPase have improved carbon assimilation, export and storage in plant source and sink organs, which results in growth, yield and quality improvements in crop plants.
Recent advances in genetic engineering have provided the prerequisite tools to transform plants to contain alien (often referred to as xe2x80x9cheterogenous or heterologousxe2x80x9d) or improved endogenous genes. The introduction of such a gene in a plant would desirably lead to an improvement of an already existing pathway in plant tissues or introduction of a novel pathway to modify desired product levels, increase metabolic efficiency, and/or save on energy cost to the cell. It is presently possible to produce plants with unique physiological and biochemical traits and characteristics of high agronomic and crop processing importance. Traits that play an essential role in plant growth and development, crop yield potential and stability, and crop quality and composition are particularly desirable targets for crop plant improvement. These improvements may be achieved by genetically modifying a crop plant for improved carbon assimilation, more efficient carbon storage, and/or increased carbon export and partitioning capabilities.
Atmospheric carbon fixation (photosynthesis) by plants, algae, and photosynthetic bacteria represents the major source of energy to support processes in such organisms. The Calvin cycle, located in the stroma of the chloroplast, is the primary pathway of carbon assimilation in higher plants. Carbon assimilates can either leave the cycle for sucrose or starch biosynthesis or continue through the cycle to regenerate the carbon acceptor molecule, ribulose-1,5-bisphosphate. Sedoheptulose-1,7-bisphosphatase is an enzyme that catalyzes an essentially irreversible reaction in the branch region where intermediates can leave the cycle, and therefore it may be essential to regulating carbon partitioning between the regeneration phase of the cycle and sucrose and starch biosynthesis.
SBPase has no known cytosolic counterpart and is reported to be found only in the chloroplast, where it dephosphorylates sedoheptulose-1,7-bisphosphate (SBP) to form sedoheptulose-7-phosphate and inorganic phosphate. This enzyme is specific for SBP and is inhibited by its products as well as glycerate (Schimkat et al., 1990) and fructose-2,6-bisphosphate (Cadet and Meunier, 1988b). Light, a reducing agent, and Mg2+ are required for activity (Woodrow, 1982; Cadet and Meunier, 1988a). The enzyme is a homodimer with a subunit molecular mass of 35-38 kDa (Nishizawa and Buchanan, 1981; Cadet and Meunier, 1988c).
It has been reported that removal of more than 80% of the enzymatic activity of SBPase in tobacco plants using antisense technology resulted in chlorosis, reduced growth rates, and reduced carbon assimilate levels (Harrison et al., 1998). Reduction in the quantum efficiency of photosystem II was also observed, which resulted in the reduction in carbohydrate content of the leaves. Analysis of carbohydrate status showed a shift from starch while sucrose levels were maintained. These results indicate that SBPase is a potential rate-limiting step in carbohydrate metabolism.
Various sedoheptulose 1,7-bisphosphatases have been characterized biochemically, and the corresponding mRNAs (cDNA) have been cloned from an alga (Genbank accession number: X74418; Hahn and Kuck, 1994) and some higher plants such as Triticum aestivum (Genbank accession number: X65540; Miles et al., 1993), Spinacia oleracea (Genbank accession number: L76556; Martin et al., 1996) and Arabidopsis thaliana (Genbank accession number: S74719; Willingham, et al., 1994). Thus, over-expression of a nucleic acid sequence encoding SBPase in a transgenic plant will provide advantageous results in the plant such as improved carbon assimilation, export and storage; increased photosynthetic capacity; and extended photosynthetic ability.
The present invention provides a method for improving the assimilation of carbon in plants using structural nucleic acid constructs that encode a sedoheptulose 1,7-bisphosphatase (SBPase) enzyme.
In accomplishing the foregoing, there is provided, in accordance with one aspect of the present invention, a method for improving the assimilation of carbon in a plant comprising the steps of:
(a) inserting into the genome of a plant a nucleic acid sequence comprising in the 5xe2x80x2 to 3xe2x80x2 direction and operably linked,
(i) a promoter that functions in the cells of a selected plant tissue,
(ii) a structural nucleic acid sequence that causes the production of a sedoheptulose 1,7-bisphosphatase enzyme,
(iii) a 3xe2x80x2 non-translated nucleic acid sequence that functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3xe2x80x2 end of a RNA sequence;
(b) obtaining transformed plant cells containing the nucleic acid of step (a); and
(c) regenerating from transformed plant cells a transformed plant that overexpresses the sedoheptulose 1,7-bisphosphatase enzyme in the plant cells.
In a further embodiment of the present invention an isolated nucleic acid sequence comprising a promoter capable of functioning in a plant cell, a structural nucleic acid sequence in sense orientation capable of causing the production of a sedoheptulose 1,7-bisphosphatase enzyme, and a 3xe2x80x2 non-translated nucleic acid sequence capable of causing transcriptional termination and the addition of polyadenylated nucleotides to the 3xe2x80x2 end of the transcribed mRNA sequence, is provided. This nucleic acid sequence may optionally include introns, 5xe2x80x2 untranslated leader sequences or other nucleic acid sequences designed to enhance transcription and/or translation.
In a still further embodiment of the invention, a novel, isolated nucleic acid sequence encoding a sedoheptulose 1,7-bisphosphatase enzyme from a green algae, Chlorella sorokiniana, is provided.
In a yet further embodiment of the present invention, a variant nucleic acid sequence encoding a sedoheptulose 1,7-bisphosphatase enzyme is provided whereby the cysteine residues in the polypeptide sequence are modified to another amino acid in a manner providing an active enzyme regardless of the presence of light.
Therefore, in accordance with the present invention, a means for improving carbon assimilation, storage and export in the source tissues of a plant is provided. Further means of improved carbon accumulation in sinks (such as roots, tubers, seeds, stems, and bulbs) are provided, thus increasing the size of various sinks (larger roots and tubers) and subsequently increasing yield. The increased carbon availability to these sinks would improve and/or alter the composition of the cellular components of the plant (e.g., oils, proteins, starch and sucrose production and solids uniformity). One aim of the present invention is to overexpress sedoheptulose 1,7-bisphosphatases in plants by introducing a heterologous source of the sedoheptulose 1,7-bisphosphatase into the plant or by increasing the expression of the endogenous form of the gene in the plant.
Various advantages may be achieved by the aims of the present invention. Increasing the expression of the sedoheptulose 1,7-bisphosphatase enzyme in the chloroplast would increase the flow of carbon through the Calvin Cycle and potentially increase atmospheric carbon assimilation in the presence of light. This would result in an increase in photosynthetic efficiency, an increase in chloroplast starch production (a leaf carbon storage form degraded during periods when photosynthesis is low or absent), and an increase in sucrose production by the leaf resulting in a net increase in carbon export to the sink and developing tissues would be expected during a given photoperiod. This increase in source capacity is a desirable trait in crop plants and would lead to increased plant growth, storage ability, yield and vigor.